Embryonic stem (ES) cells hold tremendous promise as a source of functional differentiated cell types for regenerative medicine. In addition, ES have great potential as an in vitro system for the study of developmental biology, allowing the effective isolation of distinct populations of cells that normal exist very close together in both space and time during embryonic development. Both the efficient directed differentiation of ES cells to specific lineages and studies of developmental mechanism require the in vitro recapitulation of the appropriate intermediate cell types formed in embryonic development. Thus, the successful differentiation of ES cells to neural, haematopoetic and epidermal cell types involve a progression through intermediate progenitor states prior to terminal differentiation (Bagutti et al. 1996; Nishikawa et al. 1998; Conti et al. 2005) [Conti 2005, Nishikawa 1998, Bagutti 1996]. It is likely that successful differentiation of endodermal lineages from ES cells will also depend on efficiently recapitulating the step-wise progression that occurs during normal embryogenesis.
The anterior definitive endoderm (ADE) is one the first defined lineages to emerge from the primitive streak during gastrulation. It originates in the node, the mammalian equivalent of the Spemman Organiser (also referred to as axial mesendoderm) and these cells migrate anteriorly during embryogenesis and have an important role in patterning the anterior neural axis (Lu et al. 2001). Later during gut tube formation they migrate ventrally to form the ventral foregut, a unique population of bipotent precursors of the liver and pancreas(Deutsch et al. 2001). Thus the ADE not only represents an essential signalling centre for embryogenesis, but an important intermediate in the production of liver and pancreas.
In vertebrate developmental contexts mesendoderm is often used to refer to the derivatives of the node or Spemman Organiser (here referred to as axial mesendoderm), structures that can be associated with both the mesoderm and endoderm. However, mesendoderm can also be used to refer globally to the migrating lineages at gastrulation, mesoderm and endoderm. These lineages may have evolved from a common ancestor and are induced by the same signalling pathways (Rodaway and Patient 2001), suggesting that in some instances the mesoderm and endoderm may develop from a common precursor population during embryonic development. In C.elegans, sea urchin and zebrafish, individual progenitor cells have been shown to be capable of differentiating into both mesoderm and endoderm have been identified (Rodaway et al. 1999; Angerer and Angerer 2000; Maduro et al. 2001; Davidson et al. 2002). In mouse, recent data from in vitro differentiation of ES cells provides evidence for the existence of a bi-potential mesendodermal cell in culture (Tada et al. 2005), however, whether individual epiblast derived mesendodermal progenitor exists in mouse in vivo remains to be demonstrated.
Loss of function studies in the mouse suggest that mesendoderm induction is dependent on nodal related TGF-β and canonical Wnt signalling, with the ADE being most sensitive to a reduction in the activity of these pathways (Vincent et al. 2003). This is consistent with experiments in lower vertebrates and implies that the highest activity of these two pathways is required to induce the most anterior endoderm (Green and Smith 1990; Dyson and Gurdon 1998; Chen and Schier 2001; Vincent et al. 2003). However, the identity of mesendodermal cell populations in the embryo is intricately linked to the migration of these cells during gastrulation. Thus, perturbation of these signalling pathways in the embryo may disrupt cell movements and produce a phenotype in the mesendoderm, without being directly involved in mesendoderm induction. Studying the induction of endoderm using an in vitro cell system would avoid this complication.
Several recent studies have described the generation of mesendodermal cell populations in vitro from ES cells as a starting point for the production of definitive endoderm and ventral foregut derivatives (Kubo et al. 2004; Tada et al. 2005; Yasunaga et al. 2005; D'Amour et al. 2006; Gadue et al. 2006). In mouse this work has relied on the use reporter cell lines that contain a marker under the control of a mesendodermal promoter. However, these reporter lines have been designed to identify mesendoderm precursors or all axial mesendoderm derivatives and cannot identify positionally specified populations such as the ADE. Without enrichment for ADE, the efficiency of generating foregut derivatives will be reduced. The inability to enrich for regionally specified axial mesendoderm also limits the usefulness of ES cells as an experimental system to address mechanisms of lineage specification in vitro.
It is desired to be able to provide ADE cells and then differentiate these cells into other cells, such as pancreatic and liver cells, but this is hitherto not possible in significant numbers. It is also desired to provide differentiated cells such as pancreatic or liver cells for transplantation but the persistence of ES and other non-pancreatic/non-liver cells in ES-derived cell populations can give rise to tumours in recipient animals.
A complete understanding of the molecular and cellular events controlling the behaviour of ADE cells is essential, not only as a route for understanding embryogenesis, but also as a framework upon which ADE cells can be isolated, expanded and controlled for future therapeutic and research applications. It is desirable to develop methods and conditions for culturing large quantities of ADE cells that allow the cells to be differentiated into, for example, pancreatic or liver cells. It is particularly desirable to have defined culture media, which enable this as the use of defined media is highly desirable in a clinical or research setting.
The present invention seeks to solve one or more of the above described disadvantages.
It is an object of the present invention to circumvent one or more of these problems by attempting to purify populations enriched for ADE and use these as a source of material for further differentiation.